Lactic acid bacteria have long been used as preservatives for food such as fermented milk, meat, fish, vegetables and cheese and in animal feed. Fermented foods are known to have beneficial effects on the human intestinal environment. Lactobacillus species are also useful as probiotics, microorganisms that have beneficial effects in the intestine and promote health when ingested.
Vaccines delivered orally are more convenient than the more commonly used parenteral delivery system, especially when vaccines are to be administered to large numbers of people or animals in less industrialized countries. Earlier attempts to develop oral vaccines have utilized pathogenic organisms, such as Salmonella species, as antigen carriers for oral immunization. However, even when these pathogens are attenuated they may pose a danger of reverting to pathogenicity and being harmful to the host animal. Lactic acid bacteria, in general, and Lactobacillus species in particular, possess certain properties that make them attractive candidates for use in oral vaccination. These properties of Lactobacillus include adjuvant activity, mucosal adhesive properties, and low intrinsic immunogenicity. They are generally regarded as safe (GRAS) as they are present in the animal's endogenous intestinal flora and are used commercially in the production of yogurts, cultured milks and other foods. Lactobacillus species are known to be difficult to transform with new genetic information. Those unable to be transformed are referred to as recalcitrants.
The gastrointestinal tract of animals is a complex ecosystem harboring an estimated 300 to 500 species of microorganisms. Despite over 100 years of intensive research in the field of intestinal microbiology, much remains to be learned about these microorganisms. Complex inter-relationships exist among different species of microorganisms and between resident microorganisms and their hosts.
An important factor concerning the utility of Lactobacillus species as a vaccine delivery vehicle is their ability to adhere to the epithelial cells of the animal to be vaccinated. Knowledge of the structure and mode of expression of surface related proteins of Lactobacillus that are involved in adherence to mucosal tissues and/or the extra-cellular matrix is important in designing an effective vaccination system. Adherence factors can be critical to proper antigen presentation in order for recombinant strains of lactic acid bacteria to elicit mucosal IgA and/or serum IgG responses to the expressed antigen in a host.
Lactobacilli are Gram-positive, non-sporeforming rods. They are important members of the normal human oral, gastrointestinal, and genital flora and are non-pathogenic to humans and animals. Lactobacilli including L. reuteri have been found in the gastrointestinal tract of all mammals studied to this time (Mitsuoka, 1992) including humans, pigs, chickens, cattle, dogs, mice, rats and hamsters. The ubiquity of Lactobacillus species in the mammalian gastrointestinal tract combined with their ability to target and adhere to mucosal receptors make them useful organisms as vectors for vaccinating a host against a wide range of pathogens.
Although many infectious agents gain access to the body by colonizing mucosal surfaces, very few infections caused by these agents have been effectively prevented by using mucosal, i.e., oral immunization (Wells et al, "Lactic acid bacteria as vaccine delivery vehicles", Antonie van Leeuwenhoek 70:317, Kluwer Academic Publishers, 1996). Oral immunization is highly desirable because of ease and the low cost of vaccine delivery, storage and administration. An effective delivery vehicle or organism should be one that is normally present in the gastrointestinal tract of the host organism and must accurately target the mucosal sites of infection and adhere to the mucosal surface. Lactobacilli possess both of these characteristics. A useful vaccine delivery vehicle must, in addition, be capable of expressing antigens of interest at sufficiently high levels to successfully immunize the host and must be non-pathogenic to the host.
Previous work on oral vaccination has focused on the development of modified pathogenic bacteria as antigen delivery vehicles (Stocker, U.S. Patent No. 4,837,151, Auxotrophic Mutants of Several Strains of Salmonella; Clements et al., U.S. Pat. No. 5,079,165, Avirulent Strains of Salmonella; Charles et al., U.S. Pat. No. 5,547,664, Live-attenuated Salmonella). The efficacy of these bacteria as vaccines is thought to depend on their invasiveness, capacity to survive and multiply, and on adequate levels of antigen gene expression in vivo. It is unclear, however, whether pathogenic strains that are sufficiently attenuated to pose no danger to recipients will retain their ability to invade target areas, multiply, and express adequate antigen levels (Wells et al.). This has led the present inventors to investigate the use of lactic acid bacteria, Lactobacilli and particularly L. reuteri, that have been modified to express exogenous antigens.
Leer et al. (WO095/35389) disclose a method for introducing nucleic acid into microorganisms, including microorganisms such as Lactobacillus and Bifidobacterium species that are difficult to transform or transfect. The method of Leer et al. is based on limited autolysis before the transformation process is undertaken.
Published PCT application PCT/NL96/00409 describes methods for screening non-pathogenic bacteria, in particular lactic acid bacteria of the genera Lactobacillus and Bifidobacterium, for the ability to adhere to specific mucosal receptors. The method comprises screening for adherence factors found on these non-pathogenic bacteria that are structurally related to virulence factors of some pathogenic microorganisms. An expression vector is also disclosed that comprises an expression promoter sequence, a nucleic acid sequence, and sequences permitting ribosome recognition and translation capability. This reference indicates that various strains of Lactobacillus can be transformed so as to express heterologous gene products including proteins of pathogenic bacteria.
Oral administration of recombinant L. lactis has been used to elicit local IgA and/or serum IgG antibody responses to an expressed antigen (Wells et al.). This indicates that in L. lactis, expressed heterologous proteins may elicit antigenic responses in a host organism. However, this reference and none of the prior art teaches that L. reuteri, a species with particularly desirable indigenous characteristics of mucosal targeting and adherence, can be transformed with heterologous DNA and express the foreign protein on the surface of the L. reuteri cell or secreted by the cell. The prior art fails to suggest or disclose the transformation of Lactobacillus with the aggregating gene agg or the mucin binding gene muc as set forth below.
U.S. Patent No. 5,413,960 to Dobrogosz teaches a method for obtaining the antibiotic .beta.-hydroxyproprionaldehyde, or reuterin, which is active against both Gram- positive and Gram-negative bacteria by culturing L. reuteri under anaerobic conditions in the presence of glycerol or glyceraldehyde. U.S. Pat. No. 5,352,586 also to Dobrogosz describes a method of identifying strains of L. reuteri that produce the antibiotic reuterin. In both patents the antibiotic producing L reuteri strains are identified by their ability to inhibit the growth of susceptible microorganisms in the presence of glycerol or glyceraldehyde. These references provide a method for obtaining strains of L. reuteri that secrete the antibiotic reuterin useful in the treatment of infection caused by various pathogenic microorganisms.
U.S. Pat. No. 5,439,678 claims a method for providing a probiotic to an animal which comprises feeding the animals L. reuteri. The term "probiotic" refers to ingested microorganisms that can live in a host and contribute positively to the host's health and well-being. The teachings of U.S. Pat. Nos. 5,352,586, 5,439,678 and 5,413,960 are incorporated herein by reference. These patents, however, do not suggest or disclose the use of L. reuteri as a vaccine delivery vehicle.
Heng, N.C.K. et al. (Cloning and Expression of an Endo-1,3-1,4-.beta.-Glucanase Gene from Bacillus macerans in Lactobacillus reuteri, Appl. and Environ. Microbiol, 3336-3340, Aug. 1997) describe the cloning, expression, and secretion of a heterologous gene derived from another bacterial species in a strain of L. reuteri that originated in the gastrointestinal tract. The authors believe this to be the first demonstration of the expression of a gene of heterologous origin in L. reuteri. Heng et al. were also able to demonstrate secretion by L. reuteri of the gene product, .beta.-glucanase, indicating that the heterologous secretion signals were recognized by the L. reuteri cells.